Nucleosides for imaging and treatment applications

ABSTRACT

Methods of diagnosing and/or of treating tumors by administering a nucleoside analogue which is activated by thymidylate synthase and/or thymidine kinase enzyme into a diagnostic or toxic metabolite, and uridine analogue compounds, and compositions of same having a pharmaceutically acceptable carrier. For diagnostic applications, compounds containing a label and methods of use of such compounds are described.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to methods, compounds, andcompositions for diagnosing and/or treating tumor cells with anti-tumoragents activated by thymidylate synthase (TS) and/or thymidine kinase(TK). In addition, the present invention relates to the preparation anduse of positron emitting nucleoside analogues for use in imagingapplications. The nucleoside analogues used in imaging applications maybe of the type activated by TS or, in other embodiments, may not requireactivation by TS. More particularly, the present invention relates tomethods for diagnosing and/or treating tumor cells by administration ofcompounds such as nucleoside analogue prodrugs and related compounds orcompositions containing these in an effective amount to identifysusceptible tumors in biopsy specimens or via external imaging, and thenproceeding to reduce or inhibit the replication or spread of tumorcells.

[0003] 2. Technology Review

[0004] Thymidylate synthase (TS) is an essential enzyme for DNAsynthesis. It is, however, more abundant in tumor cells than in normaltissues. For decades, research and clinical studies have been directedtowards inhibition of TS in order to shrink tumors. In some instances,this strategy has been modestly successful, for example, fluorouraciland floxuridine are utilized in the treatment of breast, colon,pancreas, stomach, ovarian, and head/neck carcinomas as disclosed by ChuE, Takimoto CH. “Antimetabolites.” In: DeVita V T Jr., Hellman S,Rosenberg S A, editors, Cancer: Principles and Practice of Oncology,Vol 1. 4th ed. Philadelphia: Lippincott, 1993:358-374.

[0005] Unfortunately, most tumors are inherently resistant to thisstrategy, and even those tumors, which are initially sensitive, developresistance during the course of treatment as reported by Swain S M,Lippman M E, Egan E F, Drake J C, Steinberg S M, Allegra C J, in“Fluorouracil and High-Dose Leucovorin in Previously Treated Patientswith Metastatic Breast Cancer,” J. Clin. Oncol, 1989;7:890-9. Recentapplications of molecular probes for TS have demonstrated a consistentrelationship between resistance and high expression of TS as noted inthe following articles: Johnston P G, Mick R, Recant W, Behan K A, DolanM E, Ratain M J, et al. “Thymidylate Synthase Expression and Response toNeoadjuvant Chemotherapy in Patients with Advanced Head and NeckCancer”, J. Natl. Cancer Inst. 1997; 89:308-13; Lenz H J, Leichman C G,Danenberg K D, Danenberg P V, Groshen S, Cohen H, Laine L, Crookes P,Silberman H, Baranda J, Garcia Y, Li J, Leichman L, “ThymidylateSynthase mRNA Level in Adenocarcinoma of the Stomach: A Predictor forPrimary Tumor Response and Overall Survival”, J. Clin. Oncol. 1996;14:176-82; Johnston P G, Lenz H J, Leichman C G, Danenberg K D, AllegraC J, Danenberg P V, Leichman L, “Thymidylate Synthase Gene and ProteinExpression Correlate and Are Associated with Response to 5-Fluorouracilin Human Colorectal and Gastric Tumors”, Cancer Res 1995; 55:1407-12;Leichman L, Lenz H J, Leichman C G, Groshen S. Danenberg K, Baranda J,et al, “Quantitation of Intratumoral Thymidylate Synthase ExpressionPredicts for Resistance to Protracted Infusion of 5-Fluorouracil andWeekly Leucovorin in Disseminated Colorectal Cancers: Preliminary Reportfrom an Ongoing Trial”, Eur. J. Cancer 1995; 31A: 1305-10. Kornmann M,Link K H, Staib L., Danenberg P V., “Quantitation of IntratumoralThymidylate Synthase Predicts Response and Resistance to Hepatic ArteryInfusion with Fluoropyrimidines in Patients with Colorectal Metastases”,Proc. AACR 38:614,1997.

[0006] A new generation of drugs designed to inhibit TS is reported byTouroutoglou N, Pazdur R. in “Thymidylate Synthase Inhibitors”, Clin.Cancer Res. 1996; 2:227-43, to be currently in final stages of clinicaltesting. Despite the enormous resources which are being expended toimprove the effectiveness of first-generation TS inhibitors, neither theexisting drugs nor this new set of compounds are effective in tumorswhich have a high level of TS activity. Presently, once a tumor hasbecome resistant due to high levels of TS, there is no specific therapyavailable.

[0007] Instead of inhibiting TS, the present inventors hypothesized thatis was possible to use this enzyme to activate uridine analogue prodrugsinto more toxic thymidine analogues. The present inventors havepreviously demonstrated in Molecular Pharmacology, 46: 1204-1209, (1994)in an article entitled, “Toxicity, Metabolism, DNA Incorporation withLack of Repair, and Lactate Production for1-2′Fluoro-2′deoxy-β-D-arabinofuranosyl)-5-iodouracil (FIAU) in U-937and MOLT-4 Cells” that 1-(2′Fluoro-2′deoxy-β-D-arabinofuranosyl)-uracil(FAU) was phosphorylated intracellularly by intact U-937 and MOLT-4cells to FAU monophosphate (FAUMP), converted to its methylated form,5-methyl-FAUMP (FMAUMP), and incorporated into DNA. These priorobservations suggested that FAU would be an appropriate prototype fortesting the cytotoxic potential of TS-activated prodrugs. It is to beunderstood that the former study produced data for different purposesand does not directly address the present discovery. To demonstrate thevalidity of the present concept, the inventors: (1) determined that TSis the enzyme which catalyzed the methylation; (2) examined the netformation rates of methylated species in a variety of cells; and (3)correlated the net formation rates of methylated species with cytotoxiceffects.

[0008] Among pyrimidine nucleosides, 2′-deoxyuridine (dUrd) analoguesare less toxic than their corresponding thymidine (dThd) analogues asindicated by Kong X B, Andreeff M, Fanucchi MP, Fox J J, Watanabe K A,Vidal P, Chou T C, in “Cell Differentiation Effects of2′-Fluoro-1-beta-D-arabinofuranosyl Pyrimidines in HL-60 Cells.” LeukRes, 1987;11:1031-9. The present inventors theorized that followingentry into the cell and phosphorylation, an analogue of dUrd would serveas a selective prodrug if TS can methylate it to generate thecorresponding dThd analogue. Thus, tumors which are resistant to TSinhibitors, because of high levels of TS, would be particularlysensitive to these deoxyuridine (dUrd) analogues, because they would bemore efficient in producing the toxic thymidine (dThd) species. Thisstrategy is completely novel, since it is entirely different from allprior approaches towards TS as an antitumor target. Contrary to previousresearch and clinical studies which are directed towards the inhibitionof TS in order to shrink tumors, the present invention utilizes TS toactivate uridine analogue prodrugs into the more toxic thymidineanalogues to reduce or inhibit tumor cells, especially tumor cells whichare inherently resistant to or develop resistance to existing therapies.The present invention is additionally highly complementary to all priorapproaches towards inhibition of TS as an antitumor target.

[0009] Further, because success of therapy with drugs such as FAU or itsanalogues is related to extent of incorporation into DNA, the analysisof DNA can provide diagnostic information regarding the optimal therapyfor a tumor. Thus, by examining a biopsy specimen of tumor, or byexternally imaging tumors, it can be predicted whether therapy with FAUor related compounds would be successful, or whether alternate therapyshould be used.

[0010] In addition to assessing tumor therapy, there are a variety ofother medical circumstances in which it is important to determine theproliferation rate (growth) of cells within a particular tissue in thebody. These include: assessment of bone marrow function (e.g., aftertransplantation and/or stimulation with growth factors), regeneration ofthe liver following surgery or injury, and expression of enzyme functionfollowing gene therapy.

[0011] Traditional approaches to determine growth rate have beeninvasive; i.e., have required obtaining a biopsy from the patient. Inaddition to the discomfort and risks associated with biopsy procedures,only a small sample of tissue is obtained. Thus, biopsies carry theinherent risk of misdiagnosis as the small sample may not berepresentative of the entire region. Thus, there is a need in the artfor other methodologies to determine the growth rate of tissues.

[0012] Non-invasive, external imaging methods avoid the need forbiopsies, and also have the capability of scanning large areas of thebody, indeed, the entire body if necessary. Since growth (proliferation)requires the synthesis of DNA from nucleosides, administration ofnucleosides which have been radiolabeled with a positron emitterprovides the ability to externally monitor events occurring within thebody by use of imaging technologies such as a PET (Positron EmissionTomograph) scanner, or other photon-detecting devices such as SPECT(Single Photon Emission Computed Tomograph), or gamma cameras.

[0013] These imaging technologies are only limited by the availabilityof probes whose biological fates provide information as to theproliferative state of the tissue examined. Thymidine is a particularlyuseful probe for monitoring growth/DNA synthesis, because it is the onlynucleoside for which direct incorporation of exogenously appliednucleoside into DNA is common by “salvage” pathways. There is nodependence upon the ribonucleotide pathways for the incorporation ofthymidine. Thymidine itself is unsuitable as a probe in these imagingtechnologies, since the molecule is rapidly degraded in the body.Analogues of thymidine such as FMAU and FIAU are excellent imagingprobes, because they: 1) completely follow thymidine pathways forincorporation into DNA; 2) are not degraded by catabolic enzymes; and 3)can be labeled with ¹⁸F, the most desirable atom for positron imaging.

[0014] Imaging probes incorporating other positron emitting moietieshave been used in the prior art. For example, a synthesis for ¹¹C-FMAUhas been reported. However, there are a number of practical limitationsdictated by the 20-minute half-life of ¹¹C. Probe molecules containing¹¹C must literally be prepared on-site and used within an hour. Thisrequirement makes it unfeasible to have a regional preparation centerand ship the molecules to surrounding medical facilities. Thus, everyfacility desiring to perform imaging studies using a ¹¹C-labeled probemust have on site the cyclotron facilities to prepare the isotope. Anadditional limitation of ¹¹C-containing labels arises when thebiological phenomena requires more than an hour for full expression. Theshort half life of ¹¹C means that insufficient ¹¹C would remain to beimaged in these situations.

[0015] In addition to ¹¹C-containing probes, probes labeled with ¹⁸F areknown in the prior art. ¹⁸F-fluorodeoxyglucose (FDG), a currentlyemployed imaging probe, is synthesized and distributed from a regionalfacility making it more easily available for imaging purposes. Further,nucleoside analogues incorporating ¹⁸F in positions other than those ofthe present invention, for example ¹⁸F at the 5 position of uracil, havebeen reported.

[0016] Notwithstanding the existence of the probe molecules discussedabove, there exists a need in the art for probe molecules for use inexternal imaging technologies. In addition, a need remains in the artfor additional therapeutic modalities for the treatment of cellproliferation disorders. These and other needs have been met by thepresent invention.

SUMMARY OF THE INVENTION

[0017] The present invention provides compounds, compositions, andmethods of diagnosing and/or treating tumors. The compounds of thepresent invention include nucleoside analogues which are activated bythymidylate synthase and/or thymidine kinase enzymes in an effectiveamount for diagnosis or to reduce or inhibit the replication or spreadof tumor cells. These compounds and compositions comprising thesecompounds are easily administered by different modes known in the artand can be given in dosages that are safe and provide tumor inhibitionat the relevant sites.

[0018] The present invention includes nucleoside analogues containing apositron emitting label moiety for use in imaging applications. Theseanalogues may be synthesized so as to require activation by TS prior toincorporation into DNA and subsequent imaging. In alternativeembodiments, the analogues of the present invention will not requireactivation by TS when used for imaging applications. In otherembodiments, the analogues may be used for imaging applications eventhough not incorporated into DNA.

[0019] Accordingly, it is the object of the present invention to providecompounds, compositions, and methods to identify susceptible tumors inbiopsy specimens or via external imaging, and/or inhibit or reduce thereplication or spread of tumor cells.

[0020] It is another object of the present invention to provide atreatment for tumors and other diseases characterized by abnormal cellproliferation by administrating these compounds or compositions eitheralone or in combination with other agents that inhibit tumor growthand/or with other classes of therapeutics used to treat such diseases.

[0021] It is another object of the present invention to assess theimpact of other treatments (e.g., by radiotherapy or other drugs) upontumor growth. In preferred embodiments, the treatments will be drugsintended to inhibit thymidylate synthase.

[0022] It is an object of the present invention to provide compounds andmethods useful for external imaging applications. In preferredembodiments, the invention includes the selection, preparation, and usesof nucleosides labeled with fluorine-18 (¹⁸F), a positron emitter. Themethods of the present invention permit treatment individualizationusing surrogate markers such as external imaging. Other embodiments ofthe invention may be useful in selecting the most effective drugs to beused against tumors in humans.

[0023] It is an object of the invention to provide a method that can beutilized to monitor and assess the efficacy of supportive treatments. Inpreferred embodiments the supportive treatments may be bone marrowtransplant and/or stimulation by growth factors. In other preferredembodiments, the present invention may be used to monitor and assess thecourse of liver regeneration after surgery or injury.

[0024] It is an object of the present invention to provide a method formonitoring the expression of genes introduced in gene therapyapplications.

[0025] Other features and advantages of the present invention will beapparent from the following description of preferred embodiments. Theseand other objects, features and advantages of the present invention willbecome apparent after a review of the following detailed description andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 depicts the general structure for many TS substrates. Forthe endogenous nucleoside, durd, W=X=Y=H, and Z=OH. FAU has only asingle substitution, W=F. A phosphate group attached to the sugar at the5′-position is also required. The corresponding thymidine analogues havea methyl group (—CH₃) at the 5-position of the uracil base.

[0027]FIG. 2 graphically depicts the effect on cell growth of 72-hrcontinuous exposure to: (A) FAU, or (B) FMAU. Cell designations:

[0028] CEM=; MOLT-4=∘; RAJI=▾; U-937=∇; K-562=▪; L1210=□.

[0029]FIG. 3 graphically depicts the association of DNA incorporationwith effect on cell growth. (A) FAU, (B) FMAU.

[0030] CEM=; MOLT-4=∘; RAJI=▾; U-937=∇; K-562=▪; L1210=□

[0031]FIG. 4 graphically depicts the relative sensitivity of cell linesto growth-inhibition by FAU compared with the activation potential forTS, measured independently as relative dehalogenation of IdUrd. The mostsensitive cell lines (U-937, CEM, MOLT-4) have 50% or moredehalogenation. The least sensitive lines (Raji, L1210) have 15% orlower.

[0032] CEM=; MOLT-4=∘; RAJI=▾; U-937=∇; K-562=▪; L1210=□

[0033]FIG. 5 graphically depicts FAUMP conversion to FMAUMP by TS inU-937 cell extracts, as demonstrated by the accumulation of tritiatedwater. The rate of conversion to FMAUMP was about 1% of the rate of dTMPformation from dUMP. Similar results were obtained for the other celllines, with a range of 0.97-1.5%.

[0034]FIG. 6 graphically depicts effect on cell growth of 72-hrcontinuous exposure to: (A) ara-U, or (B) ara-T. Cell designations:

[0035] CEM=; MOLT-4=∘; RAJI=▾; U-937=∇; K-562=▪; L121=□

[0036]FIG. 7 graphically depicts effect on cell growth of 72-hrcontinuous exposure to: (A)dUrd, or (B) dThd. Cell designations:

[0037] CEM=; MOLT-4=∘; RAJI=▾; U-937=∇; K-562=▪; L1210=□

DETAILED DESCRIPTION OF THE INVENTION

[0038] Tumor cells with high levels of thymidylate synthase (TS)represent a common therapeutic challenge for which no treatment strategyis currently available. One aspect of the present invention is that thegrowth of tumor cells with high TS can be preferentially inhibited witha uridine and/or a deoxyuridine (dUrd) analogue. As used hereinafter,uridine analogue is seen to include uridine and deoxyuridine andderivatives of both. Further, since tumors can vary widely, theidentification of tumor cells with high levels of TS provides diagnosticinformation to select appropriate therapy for individual tumors. UsingFAU as a prototype this concept has been successfully demonstrated.

[0039] The following SCHEME I illustrates the generalized structure fordUrd analogues and their intracellular activation pathways. For theendogenous nucleoside, durd, W=H. FAU has the substitution, W=F. Aphosphate group is attached to the sugar at the 5′-position by thymidinekinase (TK) to form dUMP or its analogue, FAUMP. Subsequently, TSattaches a methyl group at the 5-position of the base to generatethymidylate, dTMP, or its analogue, FMAUMP.

[0040] The present inventors demonstrated that FAU was converted intoFMAU nucleotides and incorporated as FMAU into cellular DNA. Inparticular, the monophosphate of FAU, FAUMP was S converted by TS incell extracts to the corresponding dThd form, FMAUMP. Incubation of FAUwith tumor cell lines in culture inhibited their growth to a variableextent, depending upon the efficiency of activation via TS. This is thefirst demonstration that cells with high levels of TS activity can bemore vulnerable to therapy than cells with low TS activity.

[0041] Wide variation among cell lines was observed in growthinhibition, and also relatively shallow slopes for the response versusextracellular concentration curves (FIG. 2A, 2B). As a consequence,extracellular concentration of FMAU or especially FAU was a weakpredictor of cytotoxicity, In contrast, the variation among cell linesin IC50 related to % replacement of dThd in DNA by FMAU was quite small(FIG. 3). Further, there were steep response curves for growthinhibition versus incorporation of drug (as FMAU) into DNA. Further,there was similarity among cell lines in toxicity at similar values for% replacement of dThd in DNA by FMAU. Thus, for equal exposure to theprodrug, selective toxicity could be related to differences in the rateof conversion to dThd analogues by TS. However, although conversion byTS is a necessary condition for toxicity, it is not sufficient.Opportunistic utilization of elevated TS activity also relies upon othersteps, especially kinases and polymerases, as well as competition withendogenous synthesis. Growth inhibition ultimately depends upon the netaction of all the pyrimidine pathways.

[0042] These data in FIG. 3 also demonstrate a use of deoxyuridineanalogues for diagnostic applications. Tumors with high uptake of FAUand incorporation into DNA after methylation via thymidylate synthasecould be imaged externally, e.g., by use of ¹⁸F-labeled FAU withpositron emission tomography (PET). Alternatively, a dose of FAU couldbe administered prior to a tumor biopsy, and incorporation into DNAdetermined with the same techniques used for the cell culture samples inFIG. 3. By either modality, tumors with high DNA incorporation would beexcellent candidates for therapy with FAU or related analogues, andtumors with low DNA incorporation should be treated with some othertherapy.

[0043] It is possible that FAU has autonomous biologic effects separatefrom FMAU nucleotides. However, there were several indications thatformation of FMAU by TS was sufficient to explain the majority ofobserved effects. In the present invention, comparison to the direct useof FMAU demonstrated that the toxic effects were dominated by FMAUnucleotides, especially similarity in DNA relationships. In addition,the relative sensitivity of cell lines to growth-inhibition by FAU wascompared with the activation potential for TS, measured independently asrelative dehalogenation of IdUrd (FIG. 4). The most sensitive cell lines(U-937, CEM, MOLT-4) have 50% or more dehalogenation. The leastsensitive lines (Raji, L1210) have 15% or lower dehalogenation.

[0044] Nonetheless, under other experimental conditions, if there aredifferences among cells in transport, phosphorylation, or relatedpathways, then these factors can also influence response in addition toTS activity. A major advantage of imaging tumors with labeled FAU isthat it detects the end-product of all these processes, which shouldtranslate into prognostic value.

[0045] Further, an alternative or additional approach to diagnosis issuggested by interpretation of the data in FIG. 4. Prior to biopsy, adose of labeled IdUrd, either radiolabeled or more preferably labeledwith stable isotopes, can be given to a patient. Dehalogenation can bedetermined from the DNA in the tumor biopsy and used to guide therapy.

[0046] The present invention provides a promising avenue of attack forcommon human tumors which have previously been resistant to therapeuticapproaches. Although FAU was used to demonstrate the principle, FAU wasnot very potent, and may not necessarily be the optimal compound in itsclass. The rate of methylation by TS was rather low, only 1% comparedwith the endogenous substrate, dUMP. Despite this low rate, substantialamounts of FMAU were incorporated into DNA and toxicity was observed.

[0047] If FAU is not ideal, there are many other synthetic modificationsof dUrd which can also serve as TS substrates. For example, cell culturedata were obtained for uracil arabinoside (ara-U) and its methylatedanalogue, thymine arabinoside (ara-T). As shown in FIG. 6, the patternsof toxicity for ara-U and ara-T are very similar to those for FAU andFMAU in FIG. 2, suggesting a similar mechanism, i.e., methylation.Further, the endogenous compounds, deoxyuridine (dUrd) and thymidine(dThd) also display the same pattern of cell culture toxicity (FIG. 7).

[0048] Accordingly, the present invention includes compounds,compositions and method for diagnosis and treatment of tumors. Oneembodiment of the present invention is the use of uridine analogues orrelated compounds as disclosed herein to inhibit tumor formation. Thepresent invention also includes compounds which have anti-tumoractivity. The present invention also comprises a method of treatingtumor formation in humans or animals comprising the steps ofadministering to the human or animal having tumors, a compositioncomprising an effective amount of a uridine analogue which is capable ofinhibiting tumor growth.

[0049] It is common practice to treat tumors empirically withoutdiagnostic information regarding the sensitivity of the specific tumorto a particular drug. Thus, FAU or related compounds could be useddirectly to treat tumors of a class known to have high levels of TS.Alternatively, therapy with FAU or related compounds could begin afterfailure of conventional TS inhibitors, with the inference that TS levelsare elevated. A preferred approach would use biopsy or external imaginginformation to guide therapy selection, by diagnosing which tumors wouldbe susceptible to FAU or related compounds, and which should usealternate approaches.

[0050] Tumor inhibiting and/or diagnosing compounds that can be used inaccordance with the present invention include those having the followinggeneral formula:

[0051] wherein:

[0052] A=N, C;

[0053] B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C₁-C₆), alkoxy(C₁-C₆);

[0054] D=0, S, NH2;

[0055] E=H, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkoxy, substituted alkoxy, halogen, or any substituent which is readilycleaved in the body to generate any one of the before listed groups;

[0056] G=substituted or unsubstituted cyclic sugar, substituted orunsubstituted acyclic sugar, substituted or unsubstituted mono, di, ortri-phospho-cyclic-sugar phosphate; substituted or unsubstituted mono,di, or tri-phospho-acyclic-sugar phosphate; substituted or unsubstitutedmono, di, or tri-phospho-cyclic sugar analogues, substituted orunsubstituted mono, di, or tri-phospho-acyclic sugar analogues whereinthe substituents are alkyl (C₁ to C₆), alkoxy (C₁ to C₆), halogen.

[0057] The present invention also features methods of inhibiting tumorgrowth in mammals by administering a compound according to the aboveformula in a dosage sufficient to inhibit tumor growth.

[0058] The preferred compounds are:

[0059] wherein:

[0060] A=N, C;

[0061] B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl C₁-C₆), alkoxy(C₁-C₆);

[0062] D=0, S, NH2;

[0063] E=H.

[0064] W, X, Y, Z H, hydroxy, halogen, alkyl (C₁-C₆), alkoxy (C₁-C₆), alabel containing moiety or a label;

[0065] J=C, S; and

[0066] K=O, C.

[0067] In preferred embodiments for anti-tumor activity, E may be H.

[0068] In other preferred embodiments, W is a halogen. In a mostpreferred embodiment, W is Fluorine and E is H, methyl, iodine or asubstituent readily cleaved by the body to generate one of these groups.However, other embodiments are within the scope of the presentinvention.

[0069] It is to be understood that the compounds of the presentinvention can exist as enantiomers-and that the racemic mixture ofenantiomers or the isolated enantiomers are all considered as within thescope of the present invention.

[0070] The compounds of the present invention can be provided aspharmaceutically acceptable compositions or formulations usingformulation methods known to those of ordinary skill in the art. Thesecompositions or formulations can be administered by standard routes. Ingeneral, when used to treat cell proliferative disorders, the dosage ofthe compounds will depend on the type of tumor, condition being treated,the particular compound being utilized, and other clinical factors suchas weight, condition of the human or animal, and the route ofadministration. It is to be understood that the present invention hasapplication for both human and veterinarian use.

[0071] Any of these compounds can also be used to provide diagnosticinformation regarding the tumors. For example, if a patient is having abiopsy of his/her tumor, a dose of FAU or related compound can beadministered, with or without the use of a radiolabeled atom, or morepreferably a stable isotope of the naturally occurring atom, and the DNAfrom the biopsies treated as in the cell culture experiments.

[0072] Any generally known and acceptable radioisotopes or stableisotope of a naturally occurring atom can be utilized in the presentinvention. However, ¹⁴C and ³H are preferred radioisotopes and stableisotopic labels such as ¹³C, ²H, or ¹⁵N are most preferred.

[0073] Similarly, external imaging, e.g., via positron emissiontomography (PET), can be used in particular to detect FAU or relatedcompounds labeled with ¹¹C and/or ¹⁸F. By extension from the work shownin cell culture, high levels of FAU incorporation into DNA predicts forsuccessful therapy with FAU or related compounds. Low levels of FAU inDNA suggest that an alternative therapy should be used. Thus, thepresent invention provides a method for assessing the adequacy oftreatment of tumor with various modalities, including thymidylatesynthase inhibitors, comprising administering a uridine analogue whichis labeled with a position emitter such as ¹¹C or more preferably ¹⁸Fand determining the extent of maximum TS inhibition and persistence ofTS inhibition over time between doses by external imaging preferablywith position emission tomography. These parameters can be used to guidetiming of subsequent doses or to determine when current therapy is nolonger successful and it is necessary to switch to an alternativetherapy.

[0074] In preferred embodiments for imaging applications, W may be alabel containing moiety or a label. The label may be any moiety thatpermits the detection of the nucleoside analogue. In preferredembodiments, the label includes a positron emitting atom and in a mostpreferred embodiment, W is ¹⁸F. In other preferred embodiments forimaging applications, E may be H or methyl. In a most preferredembodiment for imaging, W is ¹⁸F and E is methyl or H. However, otherembodiments are within the scope of the present invention.

[0075] It is not necessary for the nucleosides used in imagingapplications to be activated by TS. In certain embodiments, the bases ofthe nucleosides will be 5-methyl deoxyuridine analogues (i.e. thymidineanalogues). By providing the nucleoside with a methyl group at the5-position, one biosynthetic step required for incorporation of thenucleoside into DNA is eliminated. This may have the effect of speedingincorporation into the target DNA and thus providing a better imagingresults since the nucleosides should be incorporated more efficiently.In a preferred embodiment, the nucleoside used for imaging will be FMAUwherein the fluorine atom at the 2′-position will be ¹⁶F. Nucleosideshaving other modifications at the 5-position of the base may be used inimaging applications. For example, the 5-position of the base may bemodified to include an iodine atom. Thus, in a preferred embodiment, thenucleoside will be FIAU and the fluorine atom at the 2′-position will be¹⁸F. Any other modifications at the 5 position of the base may be usedin the practice of the imaging applications of the present invention. Inpreferred embodiments, the nucleoside can serve as a substrate for theenzymes required for incorporation of the nucleoside into DNA thus, thenucleoside will have a 5′-hydroxyl group that can be phosphorylated tothe nucleoside triphosphate and the resultant triphosphate can serve asa substrate for the cellular DNA polymerase enzymes.

[0076] Also, in the case of biopsy specimens, labeled IdUrd can beadministered and interpreted in terms of the cell culture data: highlevels of dehalogenation predicts for successful therapy with FAU orrelated compounds; low dehalogenation suggests that alternative therapyshould be used. Thus, the present invention also provides a method ofdiagnosing tumors which are resistant to thymidylate synthase inhibitorsby administering IdUrd which has been labeled with either a radioisotopesuch as ¹⁴C or ³H, or more preferably, with a stable isotopic label suchas ¹³C, ²H, or ¹⁵N; preparing biopsy specimens of the tumor; anddetermining the extent of dehalogenation of IdUrd by thymidylatesynthase enzymes by examination of DNA of the tumor specimens. Basedupon these results, a therapy regimen can be suggested.

[0077] For oral administration, a dosage of between approximately 0.1 to300 mg/kg/day, and preferably between approximately 0.5 and 50 mg/kg/dayis generally sufficient. The formulation may be presented in unit dosageform and may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association, the active ingredient with liquid carriers orfinely divided solid carriers or both, optionally with one or moreaccessory ingredients, and then, if necessary, shaping the product.

[0078] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients particularly mentioned herein, the formulations of thepresent invention may include other agents conventional in the art. Itshould also be understood that the compounds or pharmaceuticalcompositions of the present invention may also be administered bytopical, transdermal, oral, rectal or parenteral (for example,intravenous, subcutaneous or intramuscular) route or may be incorporatedinto biodegradable polymers allowing for the sustained release of thecompound, the polymers being implanted in the vicinity of the tumor orwhere the drug delivery is desired.

[0079]FIG. 1 shows the general structure of many uridine analogues. Thebase consists of uracil or various modifications. The interaction withTS occurs at the 5-position, where the hydrogen atom is replaced by amethyl group. The endogenous substrate for TS,2′-deoxyuridine-5′-monophosphate (dUMP), is transformed to thymidinemonophosphate (dTMP). The original class of TS inhibitors,5-fluorouracil (FUra) and 5-fluorodeoxyuridine (floxuridine, FdUrd),after intracellular conversion into FdUMP, form a ternary complex withTS and block the endogenous conversion of dUMP to dTMP. Rather thanattempting to block the 5-position as with FUra and FdUrd, the presentinvention preserves the hydrogen at the 5-position, encouraging theacceptance of the methyl donation. Thus, for those deoxyuridineanalogues which are less toxic than the corresponding thymidineanalogues, TS can increase cytotoxicity. Analogues may consist ofmodifications of the base, sugar, or both. The phosphate group at the5′-position of the sugar is usually added intracellularly (e.g., viathymidine kinase), but modified phosphate groups may be pre-formed andenter the cell intact (e.g., phosphorothiates or HPMPC).

[0080] Several modifications of the bases are feasible. The hydrogen atposition 5 and the double bond connecting carbons 5 and 6 are the mostessential requirements in the base for the most preferred TS substrate.The nitrogen at position 1 is also a preferred embodiment, however itcould be replaced by a carbon, e.g., attempting a more stable linkagewith the sugar. The hydrogen attached to N3 can also be replaced withseveral functional groups, including a halogen, acyl or alkylsubstituent. The carboxyl at C2 or C4 can be replaced with a sulfur, asin 4-thiodeoxyuridine.

[0081] A phospho-sugar (or sugar analogue) must be attached to the basein order to interact with TS. Many changes to the sugar are possiblewhile still remaining a substrate for TS. In our prototypical compound,F replaces the hydrogen atom at the 2′-position “above” the plane of thesugar (2′-F-arabino), i.e., W=F. The resulting compound, FAU, has beendemonstrated to be phosphorylated and converted to its methylated form,FMAUMP. F can also be placed below the ring at the 2′-position, X=F.Bulkier substituents at the 2′-position are synthetically possible,e.g., as reported by Verheyden JPH, Wagner D, and Moffatt JG in“Synthesis of Some Pyrimidine 2′-Amino-2¹-deoxynucleosides” in J. Org.Chem. Vol 36, pages 250-254, 1971. W=OH yields uracil arabinoside, themain circulating metabolite of ara-C. Compounds substituted in the3′-position above the ring, Y, have been synthesized, e.g., as reportedby Watanabe K A, Reichman U, Chu C K, Hollenberg D H, Fox J J in“Nucleosides. 116. 1-(beta-D-Xylofuranosyl)-5-fluorocytosines with aleaving group on the 3′ position. Potential double-barreled maskedprecursors of anticancer nucleosides” in J Med Chem 1980October;23(10):1088-1094. Below the ring at the 3′-position, Z=Fproduces an analogue of fluorothymidine, an antiretroviral agent. Asuccessful antiviral drug, 3′-thia-cytidine (3TC) is based uponreplacement of the 3′-carbon with a sulfur atom, with no substituentsattached above or below the ring. Another reported change in the sugaris the replacement of the oxygen atom with carbon, to form a carbocylicstructure, e.g., Lin T S, Zhang X H, Wang Z H, Prusoff W H, “Synthesisand Antiviral Evaluation of Carbocyclic Analogues of 2′-Azido- and2′-Amino-2′-deoxyuridine”, J Med Chem 31:484-6, 1988.

[0082] In addition to the set of single substitutions, multiplesubstitutions would also be included within the scope of the presentinvention. If both hydrogen atoms at the 2′-position are replaced withF, the resulting molecule is 2′,2′-difluoro-deoxyuridine, which is themain circulating metabolite from gemcitabine,2′,2′-difluoro-deoxycytidine.

[0083] Subsequent to methylation by TS, analogues with modifications tothe sugar at the 2′-position can be recognized by DNA polymerases andcompete with thymidine triphosphate (dTTP) for incorporation into DNA.If the 3′-OH is preserved (Z=OH), additional bases can be addedsubsequently. However, if Z is not —OH, then the analogue will serve asa terminator of chain growth. Both chain terminators (e.g., AZT) andnon-terminators (e.g., IdUrd) have biological activity, but the spectrumof effects can be quite different.

[0084] Acyclic sugar analogues such as acyclovir or cidofovir (HPMPC)have biological activity. In the case of acyclovir and related molecules(such as ganciclovir), a viral form of thymidine kinase is able tophosphorylate the drug despite the altered geometry. For HPMPC, thephosphate group is already present.

[0085] Having described the invention, the following examples are givento illustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention described.It is to be clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the present invention and/or the scope of theclaims.

EXAMPLES Materials and Methods

[0086] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

Chemicals

[0087] Non-labeled and labeled FAU, FMAU, FAUMP and dUMP were obtainedfrom Moravek Biochemicals, Brea, Calif. Radio labeled [2-¹⁴C]FAU;[³H-CH₃]FMAU, [5-³H]FAUMP, and [5-³H]dUMP had specific activities of0.056, 0.33, 11 and 2 Ci/mmol respectively. Deoxyribonuclease I (DNaseI) from bovine pancreas, Type II, and phosphodiesterase I from Crotalusatrox, Type VI, formaldehyde, tetrahydrofolate, 2′-deoxyuridine (dUrd),thymidine (dThd), uracil arabinoside (ara-U), and thymine arabinoside(ara-T) were obtained from Sigma Chemical Co., St. Louis, Mo. All otherreagents were analytical grade.

Cells

[0088] The human-derived cell lines CEM, MOLT-4, RAJI, U-937, K-562 andthe murine-derived L1210 were purchased from the American Type CultureCollection, Rockville, Md. Cells were grown and maintained as asuspension culture in RPMI 1640 medium containing L-glutamine and 10%(v/v) heat-inactivated fetal calf serum (BRL-GIBCO, Rockville, Md.).Penicillin-streptomycin solution (Sigma Chemical Co., St. Louis, Mo.)was added to achieve a final concentration of 100 units/mL and 100 Pg/mLrespectively.

Methylation of FAUMP by Thmdylate Synthase in Cell Extracts.

[0089] When TS adds a methyl group to the 5-position of dUMP to generatedTMP, the proton at that location is released. When [5-³H]dUMP is thesubstrate, TS activity in cell extracts can be assessed by monitoringthe accumulation rate of tritiated water. Here, [5-³H]FAUMP was used asthe substrate for methylation by TS, and the generation of FMAUMP wasdetermined from the release of tritiated water. Cell extracts wereprepared from each cell line by sonication of intact cells. (See:Armstrong R D, Diasio R B. “Improved Measurement of ThymidylateSynthetase Activity by a Modified Tritium-Release Assay” J. Biochem.Biophys. Methods. 1982; 6: 141-7 and Speth P A J, Kinsella T J, Chang AE, Klecker R W, Belanger K. Collins J M. “Incorporation ofIododeoxyuridine into DNA of Hepatic Metastases Versus Normal HumanLiver” Clin. Pharmacol. Ther. 1988; 4:369-75.) The methyl donor wasprovided by 5,10-methylene tetrahydrofolate, which was generated in situby the addition of formaldehyde to tetrahydrofolate. At various timesafter the addition of substrate (20AM of either [5-³H]dUMP or[5-³H]FAUMP), the reaction was stopped by addition of HCl. Unreactedsubstrate was separated from tritiated water by adsorption ontoactivated charcoal. After centrifugation, an aliquot of the supernatantwas counted for tritiated water. As shown in FIG. 5, TS in cell extractsis capable of methylating FAUMP and releasing tritiated water, albeit ata slower rate than for dUMP.

Growth Inhibition Studies

[0090] All cell lines, except for L1210, were suspended in fresh mediaat 30,000 cells/mL. L1210 cells were suspended at 10,000 cells/mL.Cells, 2 mL, were added to each of the wells of 24-well plates andincubated with either 0 to 1000 μM of FAU or 0 to 300 μM of FMAU.Incubation was conducted at 37° C. in a humidified 5% CO₂ atmosphere for72 hours. Inhibition of cellular growth was assessed by cell counting(Elzone 180, Particle Data, Inc., Elmhurst, Ill.). Under theseconditions, the control doubling times for CEM, MOLT-4, RAJI, U-937, andK-562 cells were 21-22 hours, while the doubling times for L1210 was8-10 hours.

Intracellular Nucleotide Formation and Incorporation into DNA

[0091] All cell lines, except for L1210, were resuspended in fresh mediaat 300,000 cells/mL with appropriate amount of radioactive drug. L1210cells were resuspended at 150,000 cells/iL. After 24 hours at 37° C. ina humidified 5% CO₂ atmosphere, cells were harvested for nucleotidemeasurement and DNA incorporation. Soluble nucleotides were determinedfor each cell line following exposure to 10 μM FAU. Incorporation of FAUinto DNA (as FMAU) was determined over a range of FAU concentrationsfrom 1 μM to 1 mM. As described previously by Klecker, R W, Katki A G,Collins J M. in “Toxicity, Metabolism, DNA Incorporation with Lack ofRepair, and Lactate Production for1-(2′-Fluoro-2′-deoxy-β-D-arabinofuranosyl)-5-iodouracil in U-937 andMOLT-4-cells”, Mol. Pharmacol. 1994; 46;1204-1209; and Speth P A J,Kinsella T J, Chang A E, Klecker R W, Belanger K. Collins J M. in“Incorporation of Iododeoxyuridine into DNA of Hepatic Metastases VersusNormal Human Liver.” Clin. Pharmacol. Ther. 1988; 44:369-75, DNase I andphosphodiesterase I were used to release the bases from DNA. These basesand soluble nucleotides were determined by previously reportedHPLC-based methods as noted by Klecker et al. in the previouslymentioned reference, “Toxicity, Metabolism, DNA Incorporation with Lackof Repair, and Lactate Production for1-(2′-fluoro-2′-deoxy-β-D-arabinofuranosyl)-5-iodouracil in U-937 andMOLT-4-cells.” Drug incorporation into cellular DNA was determined usingthe equation: percent incorporation=100×([drug] /([dThd]+[drug]).

[0092] Dehaloaenation as a Probe for Thymidylate Synthase Activity InSitu.

[0093] The relative activity of thymidylate synthase was determinedafter incubation of cells with 3 μM [3H]-IdUrd for 24 hours. DNA washarvested, digested, and chromatographed as described above. Some IdUrdwas incorporated into DNA with the iodine remaining intact on thepyrimidine ring. After conversion to IdUMP, part of the IdUrd wasdehalogenated by TS to dUMP, which then was converted by TS into dTMPand subsequently incorporated into DNA, and recovered in the DNA digestas [³H-dThd]. Relative TS activity in situ was defined as the fractionof IdUrd-derived material in DNA which was dehalogenated, i.e.,([³H]-dThd)/([³H]-dThd+[³H]-IdUrd). These methods could also be usedwith non-radioactive IdUrd, if a stable isotopic label is used, e.g.,¹³C, ¹⁵N, or 2H. A mass spectrometric detector would be substituted forthe radioactivity detector in the HPLC analysis. Unlabeled IdUrd can'tbe used since its dehalogenation produced unlabeled dThd, which would beindistinguishable from the endogenous pool of dThd in DNA.

Example 1

[0094] FAUMP was converted to FMAUMP by TS in cell extracts, asdemonstrated by the accumulation of tritiated water. The rate ofconversion to FMAUMP was about 1% of the rate of dTMP formation fromdUMP (FIG. 5). Continuous incubation of cells for 72 hours with the durdanalogue, FAU, produced varying degrees of growth inhibition (FIG. 2A).At 100 μM, CEM and U-937 cells were more than 50% inhibited, MOLT-4 andK-562 were somewhat less inhibited, but Raji and L1210 cells werecompletely uninhibited.

Example 2

[0095] Continuous incubation of cells for 72 hours with the thymidineanalogue, FMAU was more potent and consistently toxic. FMAU produced aconcentration-dependent inhibition of growth for all cell lines (FIG.2B). At the lowest concentration used (0.3 μM), there was a substantialeffect on the cell growth of CEM and K-562. At 100 μM all cell linesstudied were completely inhibited (>800%). The correspondingdeoxyuridine analogue, FAU, was less toxic in all cell lines and hadIC₅₀ values which were 10-fold higher than with FMAU (FIG. 2A). Moststrikingly, the growth of L1210 cells, which were very sensitive toFMAU, was not inhibited by FAU, even at 1 mM.

[0096] Both FMAU and FAU were converted intracellularly into FMAUnucleotides, and subsequently incorporated into cellular DNA asFMAU(MP). As described in Table I below, CEM and U-937 cell lines werethe most efficient cell lines at forming FMAUTP from FAU and hence FMAUwas incorporated to a higher extent into the DNA of these cell lines.TABLE I Intracellular Nucleotides Formed and Incorporation into DNA fromIncubation of Each Cell Line With 10 μM FAU for 24 Hours. FAUMP FMAUTPDNA nmol/10⁶ cells % incorp CEM 0.96 ± 0.24 2.30 ± 0.28 0.81 ± 0.10U-937 2.70 ± 0.69 1.99 ± 0.08 0.50 ± 0.02 MOLT-4 n.d. 1.92 ± 0.55 0.19 ±0.01 K-562 11.1 ± 0.9  n.d.  0.22 ± 0.002 PAJI 0.95 ± 0.17 n.d.  0.09 ±0.002 L1210 n.d. n.d.  0.08 ± 0.005

[0097] This greater DNA incorporation was reflected as increasedtoxicity noted for CEM and U-937 in FIG. 2A. In contrast, FAU producedless incorporation of FMAU into the cellular DNA of L1210 cell line.This was reflected as less than 10% decrease in growth rate, even at 1mM. K-562 cells had a noticeably higher intracellular FAUMP pool. Onlytrace amounts of FMAUMP or FMAUDP were found.

[0098] When cell growth was plotted versus % incorporation of FMAU inDNA (FIG. 3), on the same scale used for extracellular concentration,the response curve was much steeper. Further, the variation among celllines in IC50 for growth inhibition in toxicity referenced to %incorporation in DNA showed much less variation than when referenced toextracellular concentration. Full curves were obtained for FMAU in all 6cells lines. For FAU, due to the larger quantities of drug substancewhich were required, full curves were done in only 2 cell lines, andsingle points were obtained for the other cell lines.

Example 3

[0099]FIG. 4 presents the relative sensitivity of cell lines togrowth-inhibition by PAU compared with the activation potential for TS,measured independently as relative dehalogenation of IdUrd. The mostsensitive cell lines (U-937, CEM, MOLT-4) have 50% or moredehalogenation. The least sensitive lines (RAJI, L1210) have 15% orlower dehalogenation.

[0100] The toxicity of the compounds of the present invention has beenevaluated in an animal model system. FAU was administered by oral gavageto mice at a dose of 5 g/kg once per day for 14 consecutive days. Afteran additional 14 days of observation following this dosing period, theanimals were sacrificed. Histopathologic examination of murine tissuesfound no toxicity attributable to the drug treatment. Blood samples wereobtained at various times during this treatment, to confirm that thedrug was adequately absorbed. HPLC analysis of these samples indicatedFAU concentrations in plasma reached maximum levels of 750 micromolarand minimum levels of 50 micromolar.

Example 4

[0101] The present invention includes novel nucleoside analogues usefulin imaging technologies as well as methods of synthesizing suchanalogues. In preferred embodiments, the nucleoside analogue willcontain a positron emitting moiety. Such a moiety may be a single atomor a small molecule containing a positron emitting atom. In a mostpreferred embodiment, the positron emitting moiety will be an ¹⁸F atom.

[0102] The novel nucleoside analogues of the present invention may beprepared by a modification of the procedure reported by Tann C H, etal., “Fluorocarbohydrates in synthesis. An efficient synthesis of1-(2-deoxy-2-fluoro-alpha-D-arabinofuranosyl)-5-iodouracil (beta-FMAU)and 1-(2-deoxy-2-fluoro-alpha-D-arabinofuranosyl)thymine (beta-FMAU).”J. Org Chem. 50:3644-47, 1985, and by the same group in U.S. Pat. No.4,879,377 issued to Brundidge, et al.

[0103] The present method of synthesizing a compound according to thepresent invention entails contacting a first molecule of the formula

[0104] wherein R₁, R₂ and R₅ may be the same or different and areblocking groups, R₃ is a leaving group and in preferred embodiments maybe triflate, mesylate, tosylate or imidazolsulfonyl, and R₄ is H, with asecond molecule containing a label under conditions causing the transferof the label to the position occupied by R4. The resulting labeledcompound is brominated at the 1 position and then condensed with amolecule of the formula

[0105] wherein

[0106] A=N, C;

[0107] B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl (C_(l)-C₆), alkoxy(C₁-C₆);

[0108] D=O, S, NH2;

[0109] E=H, or any substituent which is readily cleaved in the body togenerate H.

[0110] The synthesis may be initiated with1,3,5-tri-O-benzoyl-alpha-D-ribofuranoside, which is commerciallyavailable, e.g., from Aldrich Chemical Company. This material ismodified to generate the precursor for fluorination by addition of animidazosulfonyl moiety at the 2-position in the ribo-(“down”) position.

[0111] 187 mg of 1,3,5-tri-O-benzoyl-alpha-D-ribofuranoside is mixedwith 1.54 mL of dry methylene chloride. The mixture is protected frommoisture with a calcium chloride or calcium sulfate drying tube whilecooling in a salt ice bath to −20° C. Slowly add 70 microliters (110 mg)of sulfuryl chloride through a dropping funnel over 20 minutes. Add 0.44mL of dry methylene chloride to wash down solids. Imidazole is added in5 equal portions totaling 10 equivalents (270 mg). Remove the reactionmixture from the cooling bath and let the reaction continue for 2 hours.As the reaction proceeds, the mixture will turn bright yellow.

[0112] After washing with water and drying with sodium sulfate,crystallize with hexane at 0-5° C. for 16 hours. The small whitecrystals are collected, dissolved in boiling acetone and filtered hot.Add boiling water, then allow to crystallize at 4° C. for 16 hours andcollect the crystals by centrifugation.

[0113] The resultant compound has been shown to be stable for at leastseveral months at room temperature, and can be shipped to the clinicalsite or regional radiosynthesis center, where it can be stored untilneeded.

[0114] Fluorination Procedure

[0115] Because of the short half-life of ¹⁸F (110 minutes), thefluorinated nucleoside must prepared on the day of its clinical use. Inthese circumstances, the reactions steps are optimized for short times,with yield as a secondary consideration.

[0116] On the day of use, 10 mg of the imidazosulfonyl sugar isdissolved in 200 microliters of acetonitrile. ¹⁸F is prepared from acyclotron in the form of KHFF₂, and 300 mCi (e.g., combined with 1.32 mgof unlabeled KHF₂) is dissolved in 50 microliters of a 1:100 dilution ofacetic acid. In the presence of various organic solvents such asdiethylene glycol or butanediol, ¹⁸F from KHF₂ displaces theimidazosulfonyl moiety on the arabinose ring, and assumes the ara-(“up”)position. The preferred reaction solvent is 200 microliters of2,3-butanediol. If a lower volume of solvent is used (more concentratedsolution of reactants), the acetic acid is not required. The preferredincubation conditions are 15 minutes at 170° C. This reaction productcan be verified with authentic material, available commercially in thenon-radioactive form from Sigma Chemical Co. A minimum of 8% yield isformed, based upon ¹⁸F incorporation.

[0117] Although imidazosulfonyl is the preferred exchanging group forfluorination, other suitable leaving groups are equivalent for thispurpose. Examples of other, suitable groups include, but are not limitedto, triflate, mesylate and tosylate can be used instead of theimidazolsulfonyl moiety. The triflate and mesylate versions are readilyfluorinated, but the reaction of the tosylate form is less satisfactory.Those skilled in the art will appreciate that other exchanging groupsare equivalent for the purposes of the present invention. So long as theexchanging group reaction with the fluorination reagent is fast,efficient and produces minimal side products, any exchanging group knownto those in the art is equivalent. Other suitable exchanging groups aredisclosed by Berridge, et al. (1986) Int. J. Rad. Appl. Inst. Part A37(8):685-693.

[0118] Alternately, we have have shown that2-fluoro-2-deoxy-1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose can beformed by direct reaction of underivatized1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose with DAST,diethylamino-sulfur trifluoride, which can be produced readily with ¹⁸F.The synthesis of ¹⁸F DAST is described by Straatmann, et al. (1977) J.Nucl. Med. 18:151-158.

[0119] At the end of the reaction period, 2 mL of methylene chloride areadded, followed by 2 mL of water. The methylene chloride layer istransferred to a tube containing 2 mL water. Then, the methylenechloride layer is transferred to another tube and dried under as streamof air or inert gas. Then, 400 microliters of acetonitrile, 100microliters of acetic acid, and 30 microliters of HBr (30w w/w in aceticacid)are added. The-reaction is conducted at 125° C. for 5 minutes,producing a minimum 500 yield of1-Br-2-F-3,5-di-O-benzoyl-alpha-D-arabinofuranose.

[0120] At the end of this reaction step, 1 mL of toluene and 0.5 mL ofwater are added. The toluene layer is transferred to another tube anddried under a stream of air or inert gas. An additional 0.5 mL tolueneis added, and dried. The bromo-fluoro-sugar is “condensed” with apyrimidine base (e.g., uracil, thymine, iodouracil) in which the 2- and4-positions have been silylated (e.g., with hexamethyldisilazane), toform bis-trimethylsilyl (TMS) derivatives. TMS-Ura is availablecommercially,from Aldrich. Other TMS-protected pyrimidine bases (e.g.,TMS-Thy, or TMS-IUra)can be prepared prior to the day of use and shippedto the site, as with the imidazosulfonyl sugar. The preparation ofTMS-protected bases is described by White, et al. (1972) J. Org. Chem.37:430. Other suitable protecting groups that can be removed after thereaction in conditions that do not cause a substantial deterioration ofthe product may be used in place of TMS. The selection of suitableprotecting groups and the conditions for their use are well known tothose skilled in the art.

[0121] 200 microliters of a solution of TMS-Ura (or other base) aredried, and 1 mL of methylene chloride is added. The mixture istransferred to the tube containing the fluoro-bromo-sugar. The tube isheated at 170° C. for 15 minutes and then dried, producing a yield of atleast 25% of 2,4-di-TMS-3′,5′-di-O-benzoyl-2′-arabino-F-2′-deoxyuridinewhen TMS-Ura is used.

[0122] To remove the blocking groups from the 3′- and 5′-positions ofthe sugar, and the 2- and 4-positions of the base, 0.3 mL of 2M ammoniain methanol is added. The mixture is heated at 130° C. for 30 minutes.The final product, e.g., ¹⁸F-FAU, is purified (e.g., using a solid-phaseor liquid extraction cartridge or high-performance liquidchromatography) and prepared for administration in any pharmaceuticallyacceptable solvent. Any solvent may be used that is safe whenadministered to a subject so long as the compound is soluble in it.Verification of the identify of the product is obtained by comparisonwith authentic nonradioactive reference material using any standardtechnique for chemical identification. For example, FAU and FIAU areavailable from Moravek Biochemicals (Brea, Calif.). FMAU also availablefrom Moravek by special order(not listed in catalog).

Example 5

[0123] The labeled nucleosides of the present invention may be used toevaluate the impact of various treatments upon tumors. Traditionally,most therapies (drugs and/or radiation) were directed towards decreasingtumor growth in a relatively non-specific fashion. More recently, anemphasis has been placed upon approaches such as differentiating thetumor to a slower-growing form and also preventing metastasis of thetumor. For both the traditional approach and newer approaches, a keyconsideration is early determination of the success or failure of theinitial treatment modality, with subsequent treatment modification asnecessary. Since all therapies have (substantial) side effects, thepenalty for incorrect assessment is two-fold: in addition to the loss ofvaluable time to find alternative treatment, needless toxicity isendured.

[0124] The standard tools for evaluation are often inadequate to providetimely information. A tumor may actually stop growing and the activemass shrink, but this success is masked by the continued presence ofnon-viable areas, such as necrotic or calcified tissue. Thus, success ofthe treatment is masked because the tumor doesn't change size byanatomically-based assessments such as X-Ray or CAT scans. Similarly,when the tumor stops responding to therapy and begins to grow, thefailure is masked initially because the viable tissue is only a minorityof the anatomically-determined lesion.

[0125] These problems can be overcome by functional imaging with thelabeled nucleosides of the present invention. Imaging methods usingcompounds of this type are more informative as they have the advantageof focusing only on the viable tissue. This permits the determination oftreatment success or failure even in the presence of the “noise” fromnonviable tissue.

[0126] To image tumors, labeled nucleosides, preferrably labeled with¹⁸F, are prepared as described above. The compounds can be generallyadministered to a subject to be imaged at a dose of from about 1 mCi toabout 60 mCi. In preferred embodiments, the labeled compounds will beadministered in doses of about 1 mCi to about 20 mCi. In a mostpreferred embodiment, the compounds of the present inventions will beadministered in doses of from about 10 mCi to about 20 mCi. The lowerlimit of the dosage range is determined by the ability to obtain usefulimages. Dosages lower than about 1 mCi may be indicated in certaininstances. The upper limit of the dosage range is determined by weighingthe potential for radiation induced harm to the subject against thepotential value of the information to be gained. In certain instances,it may be necessary to administer a dosage higher than about 60 mCi.

[0127] The radiolabeled compounds of the present invention may beadministered in any pharmaceutically acceptable solvent in which theyare soluble. In preferred embodiments, the compounds will be dissolvedin normal saline or buffered saline. The compounds of the invention maybe administered by any route known to those skilled in the art. Forexample, the administration may be oral, rectal, topical, mucosal,nasal, ophthalmic, subcutaneous, intravenous, intra-arterial,parenteral, intramuscular or by any other route calculated to deliverthe compound to the tissue to be imaged. In preferred embodiments thecompounds will be administered by intravenous bolus.

[0128] Images may be acquired from about 5 minutes after administrationuntil about 8 hours after administration. The maximum period in whichimages may be acquired is determined by three factors: the physicalhalf-life of ¹⁸F (110 minutes); the sensitivity of the detectors in theimaging machinery; and the size of the dose administered. Those skilledin the art can adjust these factors to permit the acquisition of imagesat an appropriate time. Blood samples are also generally obtained, toconfirm adequate delivery of the administered dose.

[0129] Those skilled in the art are capable of using the labeledcompounds of the present invention to obtain useful imaging data.Details on imaging procedures are well known and may be obtained innumerous references, for example, Lowe, et al. demonstrate the use ofpositron emission tomography to analyze lung nodules (J. Clin. Oncology16:1075-88, 1998) while Rinne, et al. demonstrate the use of an¹⁸F-labeled probe in imaging protocols to analyze treatment efficacy inhigh risk melanoma patients using whole-body ¹⁸F-fluorodeoxyglucosepositron emission tomography (Cancer 82:1664-71, 1998).

Example 6

[0130] The labeled nucleosides of the present invention may be used toassess bone marrow function. Blood cells which are circulatingthroughout the body have lifetimes ranging from a few days to a fewmonths. Thus, in order to sustain the circulation, blood cells arecontinuously produced by the body. The primary source of new blood cellsis from bone marrow. Normally, bone marrow function can be inferredsimply by examining peripheral blood. If the number of blood cells permL is in the normal range and remaining steady, marrow is workingsatisfactorily.

[0131] In several circumstances, marrow function may have beendestroyed, and it is desirable to rapidly and more directly assess thefunctioning of marrow. Since the production of new blood cells occursvia cell division, DNA synthesis is the key step. Thus, a labeledanalogue of thymidine, such as ¹⁸F-FMAU can be used assess marrowfunction.

[0132] One of the circumstances in which it is desirable to rapidlyobtain information on the status of the bone marrow is in conventionalanticancer chemotherapy. For many antitumor drugs, the bone marrow issubstantially damaged, which limits the amount of treatment which can betolerated by the patient. Thus, initially, blood cell counts droprapidly after chemotherapy, including loss of the key white blood cellswhich fight infection. Prolonged loss of these cells creates a seriousdanger of infections. Generally, the damage is repaired and blood countsreturn to normal levels within a few weeks. To overcome this delay inrecovery of circulating cells, growth factors (e.g., G-CSF, GM-CSF) areadministered to patients to stimulate more division among the cells inthe bone marrow. As reported by Sugawara, et al. (J. Clin. Oncology16(1):173-180, 1998), external imaging can provide evidence to show thatbone marrow cells are being stimulated. Sugawara, et al. used¹⁸F-fluorodeoxyglucose as a probe of general energy consumption toassess the state of the bone marrow cells. The use of a thymidineanalogue such as ¹⁸F-FMAU would be preferred because it more closelymonitors the key event, which is DNA synthesis.

[0133] In a more extreme circumstance, a patient's bone marrow isintentionally destroyed with radiation and chemotherapy. After thistreatment, the patient receives a “bone marrow transplant”, i.e., themarrow which was destroyed is replaced with donor marrow fromimmunologically-matched individuals, or from a supply harvested from thepatient prior to treatment. In either case, the ability of the patientto recover from treatment requires “engraftment”, i.e., that theinjected cells enter the marrow spaces and begin producing new cells toreplace those which have left the circulation. There is very widevariation in the rate at which blood counts recover, so it is criticalto determine as early as possible if the engraftment is successful. Iffailure is detected early, a second bone marrow transplant can beattempted. The longer the wait to determine if blood counts will return,the longer the period of exposure to life-threatening infections.

[0134] More recently, marrow function has been restored by transplantsfrom selected peripheral blood cells, known as stem cells. Regardless ofwhether the source of cells is the marrow or the peripheral circulation,engraftment can be monitored via ¹⁸F-FMAU and similar compounds.

[0135] The labeled nucleosides of the present invention can besubstituted into the procedure of Sugawara, et al. For example, 1-20 mCiof labeled nucleoside may be administered to a subject. Followingadministration, sequential dynamic scans may be conducted for 60 minutesfollowing injection. A number of scans of varying duration may beconducted. For example, one protocol that may be used is to conduct six10-second images, three 20-second images, two 1.5-minute images, one5-minute image, and five 10-minute images. Those skilled in the art willappreciate that other imaging protocols using varying number andduration of scans may used to practice the present invention.

Example 7

[0136] The labeled nucleosides of the present invention may be used toassess the regeneration of liver after surgery. After exposure to injury(chemical, biological, physical, including surgery), the liver is one ofthe few organs of the body which has the ability to regenerate itself.Hepatocytes begin to replicate themselves to replace lost cells. Themost essential element of the regeneration process is synthesis of newDNA molecules. A labeled analogue of thymidine, such as ¹⁸F-FMAU, wouldbe ideal for tracking the rate of regeneration. The short half-life andlower energy of ¹⁸F would be advantages compared with radio-iodine basedprobes.

[0137] Unlike most therapeutic situations, in which a baseline imagewould be compared with changes induced by treatment, no baseline istypically available in situations in which regeneration is occurring.However, the normal rate of DNA synthesis is very low, so regeneratingtissue would be readily detected. For example, Vander Borght, et al.demonstrate in rats that up to 10-fold differences in DNA synthesis canbe found in regenerating liver compared with non-regenerating liverdemonstrating the feasability of this approach. Vander Borght, et al.used radiolabeled thymidine analogues in a noninvasive measurement ofliver regeneration with positron emission tomography (Gastroenterology.1991 September; 101(3):794-9). Further, the serial evaluation of DNAsynthesis with ¹⁸F-FMAU would provide excellent information regardingthe stimulation or suppression of regeneration.

[0138] The procedure may consist of a bolus intravenous injection of¹⁸F-FMAU, although other methods of administration may be used.Typically, from about 1 mCi to about 60 mCi of labeled compound may beadministered. In preferred embodiments, about 10 mCi may beadministered. Based upon the biochemical processes within the liver andthe physical half-life of 18F, the preferred time for image acquisitionwould be 1-10 hours after the injection. Selecting the timing andduration of the scans is well within the skill of the ordinarypractitioner in the art.

Example 8

[0139] The labeled nucleosides of the present invention can be used toassess the efficacy of gene therapy applications. One currently usedmethod of gene therapy involves introducing into a cell to beeliminated, a copy of Herpes simplex virus thymidine kinase gene(HSV-tk). The presence of HSV-tk in the cell renders the cell sensitiveto the nucleoside analogue ganciclovir. In the presence of ganciclovir,cells expressing HSV-tk are killed while those not expressing the HSV-tkgene are resistant. It has previously been demonstrated by Blasberg, etal. in U.S. Pat. No. 5,703,056 that2′-fluoro-arabinofuranosyl-nucleoside analogues, FIAU in particular, arespecifically phosphorylated by HSV-tk and incorporated into the DNA ofcells expressing a functional HSV-tk. Thus, these nucleoside analoguesprovide a means of assessing the incorporation of HSV-tk into a targetcell.

[0140] The radiolabeled FIAU disclosed by Blasberg, et al. has severaldisadvantages. Most notably, radioiodine is a biohazard which persistsin the body for many days. The lower energy and shorter half-life of ¹⁸Fsubstantially reduces the biohazard. In clinical situations where thegoal is to determine what is happening at the moment, the shorthalf-life of ¹⁸F-FMAU or ¹⁸F-FIAU is a clear advantage compared withradioiodine. This permits the assessment of changes that are occurringon a daily or weekly timescale.

[0141] In addition to direct application of thymidine kinase for genetherapy, thymidine kinase can also be used as a “reporter gene” toassess whether the vector (usually a virus) entered the target cells andbecome functional. This is a particularly important strategy when thefunction of the primary gene of interest cannot be readily assessed. Inthis case, observation of thymidine kinase with ¹⁸F-FMAU or relatedcompounds is a surrogate for expression of other genes.

[0142] For assessing gene therapy applications, ¹⁸F-labeled nucleosidesare prepared as described above. After the gene therapy has beenconducted and sufficient time to permit expression of the transducedgenes, the subject can be treated with the labeled compounds of thepresent invention. The compounds can be generally administered to asubject to be imaged at a dose of from about 1 mCi to about 60 mCi. Inpreferred embodiments, the labeled compounds will be administered indoses of about 1 mCi to about 20 mCi. In a most preferred embodiment,the compounds of the present inventions will be administered in doses offrom about 10 mCi to about 20 mCi. The lower limit of the dosage rangeis determined by the ability to obtain useful images. Dosages lower thanabout 1 mCi may be indicated in certain instances. The upper limit ofthe dosage range is determined by weighing the potential for radiationinduced harm to the subject against the potential value of theinformation to be gained. In certain instances, it may be necessary toadminister a dosage higher than about 60 mCi.

[0143] The radiolabeled compounds of the present invention may beadministered in any pharmaceutically acceptable solvent in which theyare soluble. In preferred embodiments, the compounds will be dissolvedin normal saline or buffered saline. The compounds of the invention maybe administered by any route known to those skilled in the art. Forexample, the administration may be oral, rectal, topical, mucosal,nasal, ophthalmic, subcutaneous, intravenous, intra-arterial,parenteral, intramuscular or by any other route calculated to deliverthe compound to the tissue to be imaged. In preferred embodiments thecompounds will be administered by intravenous bolus.

[0144] Images may be acquired from about 5 minutes after administrationuntil about 8 hours after administration. The maximum period in whichimages may be acquired is determined by three factors: the physicalhalf-life of ¹⁸F (110 minutes); the sensitivity of the detectors in theimaging machinery; and the size of the dose administered. Those skilledin the art can adjust these factors to permit the acquisition of imagesat a an appropriate time. Blood samples may also be obtained to confirmadequate delivery of the administered dose.

[0145] Specific examples have been set forth above to aid those skilledin the art in understanding the present invention. The specific examplesare provided for illustrative purposes only and are not to be construedas limiting the scope of the present invention in any way. Allpublications, patents, and patent applications mentioned herein are eachincorporated by reference in their entirety, for all purposes.

What is claimed is:
 1. A method of treating tumors, comprising:administering a uridine analogue selected according to claim 30 in aneffective amount to reduce or inhibit replication or spread of tumorcells.
 2. A method of alleviating cytopathic effects associated withtumors, comprising administering to an afflicted patient an effectiveamount of a uridine analogue selected according to claim 30 such thatsaid cytopathic effects are alleviated.
 3. A method of treating cancer,comprising: administering a uridine analogue selected according to claim30 in an effective amount to reduce or inhibit the replication or spreadof said cancer cells.
 4. A method of diagnosing tumors which areresistant to thymidylate synthase inhibitors, comprising the steps of:(a) administering a uridine analogue selected according to claim 30prior to obtaining biopsy specimens; (b) obtaining biopsy specimens; and(c) analyzing DNA from the biopsy specimens for extent of analogueincorporation.
 5. A method of diagnosing tumors which are resistant tothymidylate synthase inhibitors, comprising the steps of: (a)administering to said tumors uridine analogue selected according toclaim 30; wherein said uridine analogue is labeled with a positronemitter; and (b) measuring DNA incorporation by external imaging.
 6. Themethod of claim 5 wherein the positron emitter is ¹⁸F or ¹¹C.
 7. Themethod of claim 5 wherein the external imaging is accomplished by usingpositron emission tomography.
 8. The method of claim 30 wherein theuridine analogue is a compound of the following general formula:

wherein: A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl,C₁-C₆),alkoxy (C₁-C₆) D=O, S, NH2; and G=substituted or unsubstitutedcyclic sugar, substituted or unsubstituted acyclic sugar, substituted orunsubstituted mono, di, or tri-phospho-cyclic-sugar phosphate;substituted or unsubstituted mono, di, or tri-phospho-acyclic-sugarphosphate; substituted or unsubstituted mono, di, or tri-phospho-cyclicsugar analogues, substituted or unsubstituted mono, di, ortri-phospho-acyclic sugar analogues wherein the substituents are alkyl(C₁-C₆) alkoxy (C₁ to C₆), halogen.
 9. A method of treating cancercomprising: administering to a cancer patient a therapeuticallyeffective amount of a composition comprising a uridine analogue selectedaccording to claim 30 and a pharmaceutically acceptable carrier.
 10. Acomposition for reducing or inhibiting the replication or spread oftumor cells, comprising: a uridine analogue selected according to claim30 and a pharmaceutically acceptable carrier.
 11. The composition ofclaim 10, wherein the uridine analogue is of the following generalformula:

wherein: A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl, C₁-C₆)alkoxy (C₁-C₆) D=O, S, NH2; and G=substituted or unsubstituted cyclicsugar, substituted or unsubstituted acyclic sugar, substituted orunsubstituted mono, di, or tri-phospho-cyclic-sugar phosphate;substituted or unsubstituted mono, di, or tri-phospho-acyclic-sugarphosphate; substituted or unsubstituted mono, di, or tri-phospho-cyclicsugar analogues, substituted or unsubstituted mono, di, ortri-phospho-acyclic sugar analogues wherein the substituents are alkyl(C₁-C₆) alkoxy (C₁ to C₆), halogen.
 12. The method of claim 1, whereinthe uridine analogue is selected form the group consisting of FAU, d-Urdand ara-U.
 13. A method of treating undesirable tumors in a human oranimal, comprising: administering to the human or animal havingundesirable tumors a composition comprising a tumor-inhibiting amount ofa uridine analogue selected according to claim
 30. 14. A method ofreducing or inhibiting replication of tumor cells, comprising the stepof: administering a therapeutically-effective amount of a uridineanalogue selected according to claim 30 alone or in combination withother pharmaceutical agents and/or other drugs used to inhibit tumorgrowth.
 15. A method for assessing the adequacy of treatment of tumorswith thymidylate synthase inhibitors, comprising the steps of: (a)administering a uridine analogue selected according to claim 30 whereinsaid uridine analogue is labeled with a positron emitter; and (b)determining an extent of maximum thymidylate synthase inhibition andpersistence of thymidylate synthase inhibition over time between dosesby external imaging.
 16. The method of claim 15 wherein the positronemitter is ¹⁸F or 11C.
 17. The method of claim 15 wherein the externalimaging is accomplished using positron emission tomography.
 18. A methodof diagnosing tumors which are resistant to thymidylate synthaseinhibitors, comprising the steps of: (a) administering to the tumorisotopically-labeled IdUrd; (b) preparing biopsy specimens of the tumor;and (c) determining the extent of dehalogenation of IdUrd by thymidylatesynthase enzyme by examination of DNA of the tumor specimens.
 19. Themethod of claim 18 wherein the isotopic label is selected from the groupconsisting of radioisotopes ¹⁴C or ³H; or stable isotopes ¹³C or ²H and15N.
 20. A compound of the formula:

wherein: A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl alkoxy(C₁-C₆) D=O, S, NH2; E=H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkoxy, substituted alkoxy, halogen, or any substituent whichis readily cleaved in the body to generate one of the before listedgroups; at least one of W, X, Y, Z is a label or a label containingmoiety having sufficient isotopic activity for imaging and the remainderof W, X, Y, Z=H, hydroxy, halogen, alkyl (C₁-C₆) substituted alkyl(C₁-C₆), alkoxy (C₁-C₆), substituted alkoxy (C₁-C₆) J=C, S; and K=O, C.21. A compound according to claim 20, wherein W is a positron emittingmoiety.
 22. A compound according to claim 21, wherein W is ¹⁸F and E isselected from the group consisting of H, methyl and iodine.
 23. A methodof synthesizing a compound according to claim 20, comprising the stepsof: contacting a first molecule of the formula

wherein R₁, R₂ and R₅ may be the same or different and are blockinggroups, R₃ is a leaving group and R₄, is H, with a second moleculecontaining a label under conditions causing the transfer of the label tothe position occupied by R₄; and contacting the resultant labeled firstmolecule with a molecule having the structure

wherein A=N, C; B=H, hydroxy, halogen, acyl (C₁-C₆), alkyl(C₁-C₆),alkoxy (C₁-C₆) D=O, S, NH2; E=H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, halogen, orany substituent which is readily cleaved in the body to generate one ofthe before listed groups.
 24. A method according to claim 23, whereinthe label is a positron emitter.
 25. A method according to claim 24,wherein the label is ¹⁸F.
 26. A method according to claim 23, whereinthe second molecule containing a label is selected from the groupconsisting of KHF₂ and DAST.
 27. A method of imaging an organism,comprising the steps of: contacting the organism to be imaged with acompound of claim 20; and imaging the organism.
 28. A method ofdetermining the proliferation rate of a tissue, comprising the steps of:contacting the tissue with a compound of claim 20; imaging the tissue;and determining the amount of the compound incorporated into the tissue,wherein the amount of the compound incorporated into the tissuecorrelates to the proliferation rate of the tissue.
 29. [CANCELED]
 30. Amethod for selecting a uridine analogue for reducing or inhibiting thereplication or spread of tumor cells, comprising the steps of: providinga uridine analogue unsubstituted in the 5-position; testing said uridineanalog for activation by thymidylate synthase; and selecting saiduridine analog when found to be activated by thymidylate synthase.
 31. Amethod according to claim 30, wherein the testing step comprises:measuring cytotoxicity of said uridine analogue with respect to at leastone cell line with a high expression of thymidylate synthase enzyme andat least one cell line with a low expression of thymidylate synthaseenzyme; and the selecting step comprises: selecting said uridineanalogue when the cytotoxicity measured with respect to the at least onecell line with a high expression of thymidylate synthase enzyme isgreater than the cytotoxicity measured with respect to the at least onecell line with a low expression of thymidylate synthase enzyme.
 32. Amethod according to claim 31, wherein the at least one cell line with ahigh expression of thymidylate synthase enzyme is selected from thegroup consisting of U937 cell line and CEM cell line.
 33. A methodaccording to claim 31, wherein the at least one cell line with a lowexpression of thymidylate synthase enzyme is selected from the groupconsisting of L1210 cell line and Raji cell line.
 34. A method accordingto claim 30, wherein the uridine analog contains a radioisotope.
 35. Amethod according to claim 34, wherein the radioisotope is ¹⁸F of ¹¹C.